The present invention relates to methods for measuring the proliferation and destruction rates of cells by measuring deoxyribonucleic acid (DNA) synthesis. In particular, the methods utilize non-radioactive stable isotope labels to endogenously label DNA synthesized through the de novo nucleotide synthesis pathway in a cell. The amount of label incorporated in the DNA is measured as an indication of cellular proliferation. Such methods do not require radioactivity or potentially toxic metabolites, and are suitable for use both in vitro and in vivo. Therefore, the invention is useful for measuring cellular proliferation and/or cellular destruction rates in humans for the diagnosis of a variety of diseases or conditions in which cellular proliferation or destruction is involved. The invention also provides methods of screening an agent for a capacity to induce or inhibit cellular proliferation or cellular destruction and methods for measuring the proliferation or destruction of T cells in a subject infected with human immunodeficiency virus (HIV).
Control of cell proliferation is important in all multicellular organisms. A number of pathologic processes, including cancer and acquired immunodeficiency syndrome (AIDS) (Ho et al., 1995, Nature 373:123-126; Wei et al., 1995, Nature 373:117-122; Adami et al., 1995, Mutat. res. 333:29-35), are characterized by failure of the normal regulation of cell turnover. Measurement of the in vivo turnover of cells would therefore have wide applications, if a suitable method were available. Prior to the present invention. direct and indirect techniques for measuring cell proliferation or destruction existed. but both types were flawed.
Direct measurement of cell proliferation generally involves the incorporation of a labeled nucleoside into genomic DNA. Examples include the tritiated thymidine (3H-dT) and bromodeoxyuridine (BrdU) methods (Waldman et al., 1991, Modern Pathol. 4:718-722; Gratzner, 1982, Science 218:474-475). These techniques are of limited applicability in humans, however, because of radiation induced DNA damage with the former (Asher et al., 1995, Leukemia and Lymphoma 19:107-119) and toxicities of nucleoside analogues (Rocha et al., 1990, Eur. J. Immunol. 20:1697-1708) with the latter.
Indirect methods have also been used in specific cases. Recent interest in CD4+ T lymphocyte turnover in AIDS, for example. has been stimulated by indirect estimates of T cell proliferation based on their rate of accumulation in the circulation following initiation of effective anti-retroviral therapy (Ho et al., 1995, Nature 373:123-126; Wei et al., 1995, Nature 373:117-122). Unfortunately, such indirect techniques, which rely on changes in pool size, are not definitive. The increase in the blood T cell pool size may reflect redistribution from other pools to blood rather than true proliferation (Sprent and Tough, 1995, Nature 375:194; Mosier, 1995, Nature 375:193-194). In the absence of direct measurements of cell proliferation, it is not possible to distinguish between these and other (Wolthers et al., 1996, Science 274:1543-1547) alternatives.
Measurement of cell proliferation is of great diagnostic value in diseases such as cancer. The objective of anti-cancer therapies is to reduce tumor cell growth, which can be determined by whether tumor DNA is being synthesized or being broken down. Currently, the efficacy of therapy, whether chemotherapy, immunologic therapy or radiation therapy, is evaluated by indirect and imprecise methods such as apparent size by x-ray of the tumor. Efficacy of therapy and rational selection of combinations of therapies could be most directly determined on the basis of an individual tumor""s biosynthetic and catabolic responsiveness to various interventions. The model used for bacterial infections in clinical medicinexe2x80x94culture the organism and determine its sensitivities to antibiotics, then select an antibiotic to which it is sensitivexe2x80x94could then be used for cancer therapy as well. However, current management practices proceed without the ability to determine directly how well the therapeutic agents are working.
A long-standing vision of oncologists is to be able to select chemotherapeutic agents the way antibiotics are chosenxe2x80x94on the basis of measured sensitivity to each drug by the tumor of the patient in question. The ability to measure cancer cell replication would place chemotherapy selection and research on an equal basis as antibiotic selection, with great potential for improved outcomes.
Accordingly, there remains a need for a generally applicable method for measuring cell proliferation that is without hazard and can be applied in the clinical arena.
The present invention relates to methods for measuring cellular proliferation and/or destruction rates by measuring DNA synthesis. In particular, it relates to the use of a non-radioactive stable isotope label to endogenously label DNA synthesized by the de novo nucleotide synthesis pathway in a cell. The label incorporated into the DNA during DNA synthesis is readily detectable by methods well known in the art. The amount of the incorporated label can be measured and calculated as an indication of cellular proliferation and destruction rates.
The invention is based, in part, on the Applicants"" discovery that DNA synthesis can be measured by labeling the deoxyribose ring with a stable isotope label through the de novo nucleotide synthesis pathway. Cellular proliferation was measured in vitro, in an animal model and in humans. In vitro, the proliferation of two cell lines in log phase growth was measured by the methods of the invention and was shown to be in close quantitative agreement with the increased number of cells by direct cell counting, which is considered the least ambiguous measure of cell proliferation. In animals, the methods of the invention were also shown to be consistent with values estimated previously by independent techniques. For example, thymus and intestinal epithelium were shown to be rapid turnover tissues, while turnover of liver cells was much slower. In humans, the observed pattern of a lag phase followed by rapid appearance of a cohort of labeled granulocytes is also consistent with previous observation.
The methods differ from conventional labeling techniques in 3 major respects. First, conventional isotopic methods label DNA through the known nucleoside salvage pathway, whereas the methods of the invention label deoxyribonucleotides in DNA by the known de novo nucleotide synthesis pathway (FIG. 1), through which purine and pyrimidine nucleotides are formed. In brief, in the de novo nucleotide synthesis pathway, ribonucleotides are formed first from small precursor molecules (e.g., glucose, glucose-6-phosphate, ribose-5-phosphate, purine and pyrimidine bases, etc.) and are subsequently reduced to deoxyribonucleotides by ribonucleotide reductase. See, e.g., FIG. 1; Reichard, 1988, Ann. Rev. Biochem. 57:349-374 (e.g., FIGS. 1 and 2); THOMAS SCOTT and MARY EAGLESON, CONCISE ENCYCLOPEDIA BIOCHEMISTRY 406-409, 501-507 (2d ed. 1988); and TEXTBOOK OF BIOCHEMISTRY WITH CLINICAL CORRELATIONS (Thomas M. Devlin ed., 3d ed. 1992)), each of which is incorporated herein in its entirety for all purposes. Through the action of ribonucleotide reductase, three deoxyribonucleotides, dADP, dCDP, and dGDP, are produced directly. These deoxyribonucleotides are then phosphorylated by nucleoside diphosphate kinase to form corresponding deoxyribonucleotide triphosphatesxe2x80x94dATP, dCTP, and dGTP. A fourth deoxyribonucleotide, dTTP, is also formed from ribonucleotide reductase, after additional remodeling. The four deoxyribonucleotide triphosphatesxe2x80x94dATP, dCTP, dGTP, and dTTPxe2x80x94are utilized to synthesize DNA. FIG. 1, which illustrates the de novo nucleotide synthesis pathway, also shows the pathway for endogenous labeling of DNA from stable isotope-labeled glucose.
Labeling via the de novo nucleotide synthesis pathway is advantageous because in most cells that enter the S-phase of the cell cycle, the key enzymes controlling de novo synthesis of deoxyribonucleotide-triphosphates (dNTP""s), in particular ribonucleotide reductase (RR), are upregulated, whereas the enzymes of the nucleoside salvage pathway (which represents an alternative pathway for formation of purine and pyrimidine nucleotides) are suppressed (Reichard, 1978, Fed. Proc. 37:9-14; Reichard, 1988, Ann Rev. Biochem. 57:349-374; Cohen et al., 1983, J. Biol. Chem. 258:12334-12340; THOMAS SCOTT and MARY EAGLESON, CONCISE ENCYCLOPEDIA BIOCHEMISTRY 543-544 (2d ed. 1988).
Second, the label can be detected in the methods of the invention in purine deoxyribonucleosides instead of pyrimidines (e.g., from 3H-dT or BrdU). This is advantageous because the de novo synthesis pathway tends to be more active for purine than pyrimidine dNTP""s (Reichard. 1978. Fed. Proc. 37:9-14; Reichard, 1988, Ann Rev. Biochem. 57:349-374; Cohen et al., 1983, J. Biol. Chem. 258:12334-12340). In fact, regulatory deoxyribonucleotides have been shown in lymphocytes (Reichard, 1978, Fed. Proc. 37:9-14; Reichard, 1988, Ann Rev. Biochem. 57:349-374) to exert negative feedback on RR for pyrimidine dNTP synthesis but positive feedback for purine dNTP synthesis, ensuring that the de novo synthesis pathway is always active for the purines but is variable for the pyrimidines.
Additionally, the methods of the invention which use stable isotope labels instead of non-stable radio-isotopes are safe for human use. Therefore, a wide variety of uses are encompassed by the invention, including, but not limited to, measurement of the rate of cellular proliferation and/or destruction in conditions where such information is of diagnostic value, such as cancer, AIDS, hematologic disorders, endocrine disorders, bone disorders and organ failure. Where non-toxic stable isotopes are employed, such cellular proliferation and destruction rates can be measured in vivo in a subject.
In one aspect, the invention provides methods for measuring cellular proliferation or cellular destruction rates which comprise contacting a cell with a detectable amount of a stable isotope label which is incorporated into DNA via the de novo nucleotide synthesis pathway, and detecting the label in the DNA.
The invention also provides methods for measuring the rates of cellular proliferation and/or cellular destruction in a subject. Such methods comprise contacting a cell with a detectable amount of a stable isotope label which is incorporated into DNA via the de novo nucleotide synthesis pathway and detecting the label in the DNA of the subject.
In another aspect of the invention, methods for measuring the rates of proliferation and/or destruction of T cells in a subject infected with human immunodeficiency virus (HIV) are provided. Such methods comprise administering a detectable amount of a stable isotope label to the subject, wherein the label is incorporated into DNA of the T cells of the subject via the de novo nucleotide synthesis pathway. The label in the DNA of the T cells of the subject is detected to measure the rates of proliferation and/or destruction of T cells in the subject.
The invention also provides methods of screening an agent for a capacity to induce or inhibit cellular proliferation. Such methods comprise contacting a cell with the agent, contacting the cell with a detectable amount of a stable isotope label which is incorporated into DNA of the cell via the de novo nucleotide synthesis pathway, and detecting the label in the DNA. The amount of label, compared to a control application in which the cell is not exposed to the agent, indicates the extent of cellular proliferation and thereby whether the agent induces or inhibits cellular proliferation.
In another aspect, the invention provides methods of screening an agent for a capacity to induce or inhibit cellular proliferation in a subject exposed to the agent. Such methods comprise exposing the subject to the agent; administering a detectable amount of a stable isotope label to the subject, wherein the label is incorporated into DNA of the subject via de novo nucleotide synthesis pathway; and detecting the label in the DNA of a cell of interest in the subject indicating cellular proliferation in the subject. The amount of label relative to a control application in which the subject is not exposed to the agent indicates the extent of cellular proliferation and thereby whether the agent induces or inhibits cellular proliferation in the subject.
In yet another aspect of the invention, methods for measuring cellular proliferation in a proliferating or dividing population of cells are provided. These methods comprise: (a) contacting the proliferating population of cells with a detectable amount of a first label. wherein the first label comprises a stable isotope label which is incorporated into DNA via the de novo nucleotide synthesis pathway; (b) detecting the first label incorporated into the DNA to measure cellular proliferation in the proliferating population of cells; (c) contacting the proliferating population of cells with a detectable amount of a second label, wherein the second label comprises a radioactive isotope label which is incorporated into DNA via the de novo nucleotide synthesis pathway; and (d) detecting the second label incorporated the DNA to measure cellular proliferation in the proliferating population of cells. In some such methods employing both radioactive and non-radioactive isotope labels, steps (a) and (b) are performed before steps (c) and (d). In other such methods utilizing both radioactive and non-radioactive isotope labels, steps (c) and (d) are performed before steps (a) and (b). Alternatively, in some such methods, steps (a) and (c) can be performed simultaneously and steps (b) and (d) can be performed simultaneously.
The present invention also includes methods for determining the susceptibility of a subject to a disease or disorder (including disorders which are not yet themselves disease, but which predispose the subject to a disease) which induces a change in a rate of cellular proliferation in the subject. Such methods comprise exposing the subject to a condition or an agent which can produce or induce the disease or disorder and administering a detectable amount of a stable isotope label to the subject. The label is incorporated into DNA of the subject via de novo nucleotide synthesis pathway. The label in the DNA of the subject is detected. An increase in label in the DNA of the subject, compared to a control application in which the subject is not exposed to the condition or agent. indicates an increase in the rate of cellular proliferation and susceptibility of the subject to the disease or disorder.
The invention also includes methods for determining the susceptibility of a subject to a disease which induces a change in a rate of cellular destruction (including disorders which are not yet themselves disease, but which predispose the subject to a disease) in a subject. These methods comprise exposing the subject to a condition or an agent which can produce the disease, administering a detectable amount of a stable isotope label to the subject, which label is incorporated into DNA of the subject via de novo nucleotide synthesis pathway, and detecting the label in the DNA of the subject. A loss of label in the DNA of the subject compared to a control application in which the subject is not exposed to the condition or agent indicates an increase in the rate of cellular destruction and susceptibility of the subject to the disease.
In another aspect, the invention provides methods of labeling DNA in a cell which comprise contacting the cell with a detectable amount of a stable isotope label which is incorporated into DNA via de novo nucleotide synthesis pathway.